| Objective: The h BMP-2 gene was transfected into ADSCs with adenovirus,some of which was then injected into the osteoporotic fracture model for observation of its influence on fracture recovery and bone mass.Methods:(1)The ADSCs were isolated and extracted from the adipose tissue of New Zealand Rabbits by "non-enzymatic method".After subculture,the third generation ADSCs were selected,then the specific surface antigen:CD44,CD49 d,CD34 and CD106 were analyzed by flow cytometry.The osteogenic differentiation ability of the third generation ADSCs were further identified by alizarin red staining after osteogenic induction.(2)The recombinant adenovirus vectors(Ad-h BMP-2/GFP)were amplified by HEK293 cells.After repeated infection,freezing and thawing and collection,a high concentration virus liquid could be obtained.The purified recombinant adenovirus vectors infected the third generation ADSCs with MOI 100,then the transfection efficiency was detected by fluorescence microscope.The successfully transfected ADSCs were cultured for 10 days,and the expression of h BMP-2 m RNA was detected by RT-PCR,the expression of h BMP-2 protein in transfected ADSCs was detected by immunofluorescence.(3)60 successfully established osteoporosis fracture rabbits were selected,which were randomly divided into 3 groups.The experimental group were locally injected the transfected ADSCs,in the same way,the 2 control groups were injected non transfected ADSCs and saline separately.All rabbits were sacrificed,2,4 and 7weeks after injection,and the injected site of fracture were examined by X-ray and dual-energy x-ray absorptiometry scans.Objective to investigate the effect of transfected adipose derived stem cells on the repair of osteoporotic fractures.Results:(1)After 72 hours' culture,the appearance of fibroblast like cells and their rapid proliferation could be observed.On the sixth day,the cells covered the bottom of the bottle,and they had the morphological characteristics of mesenchymal stem cells.More than 80% cells whoes surface antigen-CD44 and CD49 d performed positive.Nevertheless,the expression of CD34 and CD106 performed negative.Alizarin red staining performed that a number of red stained nodules were observed under microscope when the F3 were cultured to Day 21.The results showed that ADSCs was obtained with high purity.(2)Ad-h BMP-2/GFP infected the HEK 293 cells by MOI100,which can appear completely CEP phenomenon after 3 days.Collection and lysis of the infected HEK-293 cells to obtain the amplified recombinant adenovirus.The adenovirus can reach a high titer: about 5 x 108 PFU/ml.The purified Ad-h BMP-2/GFP was transfected into ADSCs by MOI 100 and the transfection efficiency was detected.The expression of GFP reached the peak on the 6th day,and the transfection rate reached about 85%.The expression of BMP-2 m RNA was detected by RT-PCR.The BMP-2 c DNA fragment of 440 bp was found in the transfected group.No BMP-2c DNA specific band was observed in the untransfected group.On the 7th day of Immunohistochemistry,cells of h BMP-2 gene transfected ADSCs were significantly h BMP-2 protein expression.(3)Three groups of animals were examined by X-ray and dual-energy x-ray absorptiometry scans at week 2,4 and 7,respectively.The fracture healing X-ray scores of the transfected group and the non-transfected group were higher than blank control group(P< 0.05),and the differences were statistically significant.The scores of the transfected group were higher than those of the non-transfected group(P< 0.05).The result of the comparison between groups at each time is almost the same.2 weeks after injection,the callus density of each group increased,but the Blank control group was significantly lower than the other two groups(P< 0.05).In addition,the transfected group is higher than the non-transfected group,the difference was statistically significant(P< 0.05).At week 4,three groups of callus bone mineral density were further increased,the trend lasted for the second week(P< 0.05).At week 7,the callus density of the transfected group was significantly lower than that of the other two groups,and the blank control group was higher than the non-transfected group(P< 0.05).Conclusion:(1)High purity ADSCs could be extracted by the "non-enzymatic method",.(2)ADSCs successfully transfected by h BMP-2 gene,which could highly express BMP-2 protein.(3)ADSCs transfected by h BMP-2 gene could significantly improve the healing of osteoporotic fracture in rabbits. |
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